J Appl Physiol 118: 1229–1233, 2015. First published April 2, 2015; doi:10.1152/japplphysiol.00865.2014.

Plasma arginine metabolites reflect airway dysfunction in a murine model of allergic airway inflammation Jeremy A. Scott,1,2,3 Michelle L. North,2 Mahrouk Rafii,4 Hailu Huang,4 Paul Pencharz,4,6 and Hartmut Grasemann2,4,5,6 1

Department of Health Sciences, Faculty of Health and Behavioural Sciences, Lakehead University, and Division of Medical Sciences, Northern Ontario School of Medicine, Thunder Bay, Ontario, Canada; 2Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 3Dalla Lana School of Public Health, Division of Occupational and Environmental Health, University of Toronto, Toronto, Ontario, Canada; 4Program in Physiology and Experimental Medicine, Research Institute, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario Canada; 5Division of Respiratory Medicine, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario Canada; and 6Department of Pediatrics, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario Canada Submitted 25 September 2014; accepted in final form 31 March 2015

Scott JA, North ML, Rafii M, Huang H, Pencharz P, Grasemann H. Plasma arginine metabolites reflect airway dysfunction in a murine model of allergic airway inflammation. J Appl Physiol 118: 1229–1233, 2015. First published April 2, 2015; doi:10.1152/japplphysiol.00865.2014.—L-Arginine metabolism is important in the maintenance of airway tone. Shift of metabolism from the nitric oxide synthase to arginase pathways contributes to the increased airway responsiveness in asthma. We tested the hypothesis that systemic levels of L-arginine metabolites are biomarkers reflective of airway dysfunction. We used a mouse model of acute allergic airway inflammation to OVA that manifests with significant airway hyperresponsiveness to methacholine. To determine tissue arginase activity in vivo, the isotopic enrichment of an infused L-arginine stable isotope and its product amino acid L-ornithine were measured in lung and airway homogenates using liquid chromatography-tandem mass spectrometry. Tissue and plasma concentrations of other L-arginine metabolites, including L-citrulline and symmetric and asymmetric dimethylarginine, were measured and correlated with lung arginase activity and methacholine responsiveness of the airways. The effectiveness of intratracheal instillation of an arginase inhibitor (boronoethylcysteine) on pulmonary arginase activity and circulating concentrations of L-arginine metabolites was also studied. We demonstrate that 1) plasma indexes of L-arginine bioavailability and impairment of nitric oxide synthase function correlate with airway responsiveness to methacholine; 2) plasma levels of L-ornithine predict in vivo pulmonary arginase activity and airway function; and 3) acute arginase inhibition reduces in vivo pulmonary arginase activity to control levels and normalizes plasma L-ornithine, but not L-arginine, bioavailability in this model. We conclude that plasma L-ornithine may be useful as a systemic biomarker to predict responses to therapeutic interventions targeting airway arginase in asthma. arginine metabolism; airway function; biomarker NITRIC OXIDE (NO) CONTRIBUTES

to the homeostatic regulation of bronchomotor tone in the airways (20). The balance between metabolism of L-arginine via the NO synthase (NOS) and arginase pathways is critical to the maintenance of airway tone. Reports by us and others have shown that arginase expression and activity are increased in asthma (16, 19, 31), mainly in epithelial cells lining the airways and macrophages (17), and may contribute to the development of airway hyperresponsiveness, independent of its competition for substrate (18). ArgiAddress for reprint requests and other correspondence: H. Grasemann, The Hospital For Sick Children, 555 Univ. Ave., Toronto, ON, Canada M5G 1X8 (e-mail: [email protected]). http://www.jappl.org

nase inhibition, therefore, appears to be an attractive target for future therapeutic interventions to treat asthma (6). In addition, accumulation of the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), is important in NO-mediated airway reactivity in asthma, as it contributes to the shift of L-arginine toward arginase metabolism (19, 23). Arginase activity has previously been reported to be increased in serum of humans with acute asthma, whereas arginine levels were decreased (16). Arginine bioavailability measured in plasma, typically determined as the ratio of L-arginine to L-ornithine or L-arginine to L-ornithine ⫹ L-citrulline (8, 29), was found to be greater in asthma patients than controls and was related to airflow obstruction in patients with severe asthma, but not with the fraction of exhaled NO or other markers of inflammation (14). Furthermore, the ratio of L-arginine to ADMA in plasma, which can be used as an index of impaired NOS function (23), was recently shown to correlate with respiratory symptoms, worse asthma quality of life, and pulmonary function in lateonset asthma (10). Circulating markers of pulmonary arginine metabolism may, therefore, represent pathological changes in airway function in asthma. Recent studies have shown that expectorated sputum and exhaled breath condensate can be used to demonstrate alterations in the L-arginine metabolism in asthma airways (4, 21-23), but the degree to which peripheral blood changes in L-arginine metabolites reflect changes in arginase activity or arginine bioavailability in the lungs and airways remains to be determined. The aim of the present investigation, therefore, was to test the hypothesis that changes in arginase activity reflect the functional outcomes in a mouse model of allergic airway inflammation and dysfunction and to determine whether arginine metabolites in plasma reflect changes of L-arginine metabolism in the lung. We used L-arginine stable isotope infusion to measure arginase activity in vivo in lung, trachea, and plasma. We then investigated whether isotopic enrichment of L-ornithine from L-arginine or plasma concentrations of the L-arginine metabolites L-arginine, L-ornithine, L-citrulline, its derivatives ADMA, and symmetric dimethylarginine (SDMA), or indexes of L-arginine bioavailability (L-arginine/L-ornithine) and impaired NOS function (L-arginine/ADMA) reflect functional changes in the lung in this mouse model. Finally, we show that intratracheal instillation of the arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), which our laboratory has

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Plasma Arginine-Metabolites Reflect Lung Function

RESULTS

In vivo arginase activities. Arginase activity in vivo, expressed as L-ornithine enrichment from L-arginine (MPE Arg ¡ Orn) in the lung and trachea extracts and plasma samples from the OVA model mice are shown in Fig. 1. Arginase activity was augmented 2.75-, 2.9-, and 3.5-fold in lung, trachea, and plasma, respectively, from OVA/OVA mice, com-

Lung

Arginine

Ornithine

A

90 80 70 60 50 40 30 20 10 0

*

OVA/PBS

OVA/OVA

Plasma

B

175

Ornithine

Murine model of allergic airway inflammation and pulmonary function testing. This acute murine model of allergic airway inflammation was approved by the University of Toronto Faculty Advisory Committee on Animal Services and was conducted in accordance with the guidelines of the Canadian Council on Animal Care. Briefly, female BALB/c mice (8-wk old) were sensitized to ovalbumin (OVA) on days 0 and 7 and challenged with aerosolized OVA (OVA/OVA) or PBS (OVA/PBS) for 25 min/day for 7 consecutive days (days 14 –20) (5, 17, 23). On day 21, 24 h after the final aerosol challenge, mice were anesthetized (ketamine/xylazine) for tracheal and jugular venous cannulations for ventilator-based measurement of respiratory function and infusion of the stable L-arginine isotopomer cocktail, respectively. The linear first-order single-compartment model and the constant-phase model of respiratory mechanics were used to assess total respiratory and central airway and peripheral lung function, respectively (9, 24). During the pulmonary function testing, ventilation was set at a rate of 150 breaths/min and a tidal volume of 8 ml/kg, with a positive end-expiratory pressure of 3 cmH2O maintained throughout. All data were collected using the flexiVent (SciReq, Montréal, Quebec, Canada). Stable isotopes were infused after determination of baseline total lung resistance, central airway resistance, and peripheral tissue damping. Stable isotope experiments and liquid chromatography-tandem MS. All mice received a primed and constant infusion of a solution containing L-arginine (m⫹6 L-arginine U-13C6; priming dose: 850 nmol, constant infusion: 1,700 nmol/h) and L-ornithine (m⫹2 L-ornithine 15N2; priming dose: 215, constant infusion: 430 nmol/h) (Cambridge Isotope Laboratories) in normal saline (0.9%) via the jugular vein, similar to that previously reported by our laboratory (7, 11). Blood samples were collected via the tail vein at baseline (before) and after 45 min of infusion and were subsequently processed for the isolation of plasma. After assessment of respiratory and airway responsiveness to methacholine, mice were euthanized with ketaminexylazine, and necropsy was performed to isolate the lung and trachea for subsequent mass spectrometric (MS) analysis. A subset of mice were treated with intratracheal instillation of the arginase inhibitor BEC (40 ␮g/g body wt; Alexis Biochemicals, San Diego, CA) after cannulation, but before infusion of the isotope cocktail and assessment of methacholine responsiveness, as described previously (19). Tissues (lung and trachea) were homogenized in 2.5 ml, 0.1% formic acid and 11.25 ml methanol (MeOH) per gram tissue. After centrifugation, the supernatants were stored at ⫺80°C for MS analysis. Aliquots (250 ␮l) of the frozen tissue supernatant were dried under nitrogen (N2). The dried amino acids from the lung and trachea supernatants were reconstituted in 250 and 150 ␮l, respectively, of 10 mM ammonium acetate (NH4Ac; pH ⫽ 4.1) for subsequent MS analysis. Aliquots (50 ␮l) of plasma were deproteinated with 500 ␮l of MeOH. After centrifugation, the supernatants were dried under N2. The dried amino acids were reconstituted in 250 ␮l, 10 mM NH4Ac, pH ⫽ 4.1. Each of the infused isotopomers and its product amino acids were measured in serum and organ tissue homogenates using liquid chromatography-tandem MS, as described previously (25, 26). The isotopic enrichment was determined using previously described formulas (26, 28). Ornithine enrichment from arginine was expressed as moles percent excess (or MPE Arg ¡ Orn) and was used to express tissue arginase activity (7, 11). In a subgroup of animals that did not undergo infusion of stable isotopes, liquid chromatography-tandem MS was also used to quantify L-arginine, L-ornithine, L-citrulline, ADMA, and SDMA in lung, trachea, and serum, as previously reported (7, 11, 15, 23).

Scott JA et al.

Statistical analyses. All data are expressed as the means ⫾ SE. Binary comparisons were made using two-tailed unpaired Student’s t-test or Mann-Whitney U-test, where appropriate. Correlations between the biochemical parameters and functional end-points (i.e., respiratory system or airway resistance) were determined by Spearman’s test. P values ⬍0.05 were considered significant. All statistical analyses were conduced using GraphPad Prism 4.0c (Graphpad Software, La Jolla, CA).

150

Arginine

MATERIALS AND METHODS



**

125 100 75 50 25 0

OVA/PBS

OVA/OVA

Trachea

C

70

Ornithine

shown previously to restore airway contractility to normal (19), restores pulmonary arginase activity and circulating L-ornithine levels to that observed in controls.

60

Arginine

1230

**

50 40 30 20 10 0

OVA/PBS

OVA/OVA

Fig. 1. L-Ornithine enrichment (moles percent excess) in lung (A), plasma (B), and trachea (C) from ovalbumin (OVA)/PBS and OVA/OVA mice. Values are means ⫾ SE; n ⫽ 9 and 7 samples from OVA/PBS and OVA/OVA mice, respectively. *P ⬍ 0.05 and **P ⬍ 0.005 vs. OVA/PBS.

J Appl Physiol • doi:10.1152/japplphysiol.00865.2014 • www.jappl.org

Plasma Arginine-Metabolites Reflect Lung Function

Table 1. Plasma concentrations of L-arginine metabolites and endogenous NOS inhibitors (ADMA, SDMA) and indexes of L-arginine bioavailability and NOS impairment from OVA/PBS- and OVA/OVA-treated mice Metabolite L-Arginine,

nmol/mg

protein L-Ornithine,

OVA/PBS

34.8 ⫾ 6.5

OVA/OVA

21.7 ⫾ 6.7

Fold Change P Value

⫺0.4

NS

nmol/mg

protein 148.3 ⫾ 11.5 184.4 ⫾ 10.8‡ 1.2 L-Citrulline, nmol/mg protein 53.0 ⫾ 3.1 52.1 ⫾ 2.2 ADMA, nmol/mg protein 0.71 ⫾ 0.03 0.87 ⫾ 0.06‡ 1.2 SDMA, nmol/mg protein 0.141 ⫾ 0.006 0.208 ⫾ 0.003‡ 1.5 L-Arginine bioavailability* 0.18 ⫾ 0.04 0.05 ⫾ 0.02 ⫺0.7 NOS impairment† 46.9 ⫾ 8.4 14.2 ⫾ 4.6 ⫺0.7

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Scott JA et al.

Arginine

Ornithine

15

10

5

0 125

150

175

200

225

250

Plasma [L-ornithine] ( M) Fig. 2. Plasma L-ornithine concentrations reflect pulmonary (), but not tracheal (Œ) or plasma () arginase, activities in OVA/OVA mice. Spearman rs ⫽ 0.786, 0.143, and 0.252, respectively. n ⫽ 7, 8, and 8 for lung, trachea, and plasma, respectively. P ⫽ 0.048, 0.782, and 0.658, respectively.

A

2.5

RN (cmH2O.s/mL)

previously been shown by our laboratory to significantly reduce airway RN to methacholine in OVA mice (17–19), resulted in restoration of both pulmonary arginase activity and plasma L-ornithine levels to control levels (pulmonary arginase

2.0

rs= -0.58, P

Plasma arginine metabolites reflect airway dysfunction in a murine model of allergic airway inflammation.

L-arginine metabolism is important in the maintenance of airway tone. Shift of metabolism from the nitric oxide synthase to arginase pathways contribu...
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